1. Field of the Invention
The present invention relates to a method and assay kit for the analysis of the oxidative modification of protein-containing substances and of the oxidative stress in biological samples by measuring the antiradical properties of their protein-containing components.
2. Discussion of the Background
Oxidative stress is a common phenomenon which is implicated in the etiopathogenesis of several diseases such as atherosclerosis, cancer, acute inflammation, etc. Methods determining the concentration of species reactive with thiobarbituric acid (TBA) or of conjugated dienes have been used as routine measurements for the determination of the degree of severity of oxidative stress. Products of lipid peroxidation, for example, malondialdehyde and 4-hydroxynonenal are reactive with thiobarbituric acid.
One drawback of the known methods for the determination of oxidative stress is the lack of specificity, because several substances react with thiobarbituric acid. Another drawback is the relative insensitivity because lipid peroxidation does not immediately accompany oxidative stress. In fact, lipid peroxidation occurs only after antioxidants have been exhausted (see FAVIER, A.: Oxidative stress: value of its demonstration in medical biology and problems posed by the choice of a marker, Ann. Biol. Clin. (Paris), Vol. 55, 1997, pp. 9–16).
Proteins, unlike lipids, react immediately to oxidative stress. Different alterations, particularly in amino acids are detected in protein degradation assays (see PACIFICI, Robert E.; DAVIES, Kelvin J. A.: Protein Degradation as an Index of Oxidative Stress. In: METHODS IN ENZYMOLOGY, Vol. 186, Part B, Eds. Packer, L. and Glazer, A. N. Academic Press, Inc. 1990, pp. 485–502). These alterations include formation of characteristic products, alterations in the secondary, tertiary and quaternary structure, electric charge, folding, hydrophobicity, fragmentation, covalent inter- and intramolecular cross-linkage or increase in proteolytic sensitivity. However, determination of these parameters is very complicated, expensive, cumbersome and often non-specific, requiring methods such as radioactive or fluorescent labeling, gel electrophoresis, Western blots and immuno-precipitation.
Accordingly, there has been a need for a simplified method that allows determination of oxidative stress in organisms and the evaluation of antiradical activity of substances, particularly without the interference of low-molecular weight antioxidants, such as ascorbic acid and uric acid.